Project summary Nucleotide sugar transporters (NSTs) are a family of proteins that are a critical part of the glycosylation machinery in all eukaryotes as they are responsible for transporting nucleotide sugars from the cytoplasm, where they are synthesized, into the Golgi lumen where they are then utilized by glycosyltransferases to glycosylate proteins and lipids. NSTs provide an additional essential role of transporting the nucleotide monophosphate byproduct of the glycosyltransferase reaction, which is inhibitory towards glycosyltransferases, back to the cytoplasm where it can be recycled. Since NST activity controls the concentrations of both nucleotide sugars and nucleotide monophosphates in the Golgi lumen, disruption of NST activity can have many adverse physiological effects as is seen in a number of diseases caused by either mutations in NST genes or dysregulation of NST activity. However, selective inhibition of NSTs also has the potential to be exploited for therapeutic benefit in targeting parasites that depend on particular glycoconjugates for virulence as well as in blocking certain glycosylation patterns that promote tumor metastasis. Our long-term goal is to understand the molecular details that underlie substrate recognition, substrate selectivity, and the mechanism of transport of NSTs. Although NSTs were first described nearly four decades ago, many of these questions remain largely unanswered, primarily due to a lack of structural information for NSTs as well as limited methods for biochemical characterization of this family of proteins. To overcome these obstacles, we have developed methods to express, purify, and crystallize a mammalian NST, the mouse CMP-sialic acid transporter (CST). We have also developed novel binding and transport assays that will allow us to address questions regarding substrate recognition and selectivity. These methods will allow us to determine high- resolution structures of CST in complex with its substrates CMP and CMP-sialic acid, as well as structures that represent different states of the transport cycle. These structures combined with biochemical studies will answer the fundamental questions regarding the structure-function relationship of NSTs, which will further our understanding of the role NSTs play in physiology and aid in the development of drugs to target NSTs.